Ratiometric fluorescence imaging is an important tool for investigation of biological function in living cells. Confocal microscopy allows three-dimensional exploration of cell function and structure. This project will develop a novel optical attachment that will permit video- rate CCD ratio imaging on the InSight line-scanning confocal microscope, offered by Meridian Instruments, Inc. For a given frame period (i.e. 33 msec) and detector sensitivity level, a line-scanning confocal allows a higher signal to noise ratio than a point scanner, because it detects fluorescence at all points along the line at the same time, increasing the dwell time allowed during the frame period. This advantage allows real-time confocal-imaging of biological structures that are often fluorescence-limiting for a point scanner. In Phase I, an optical apparatus will be developed to split and filter two fluorescent wavelengths from the InSight image, and to deliver them side-by-side to a CCD camera. Appropriate cameras will be selected and software developed to accomplish 30, or more, ratioed images per second (488 x 256 pixels). The prototype instrument will allow exploration of real- time, confocal ratio imaging as well as real-time co- localization studies of biological structures. In Phase II, a fully integrated real-time ratio confocal microscope will be developed. PROPOSED COMMERCIAL APPLICATION: The availability of a video-rate ratio imaging on the InSight confocal microscope would greatly broaden the number of live cell applications for which the instrument could be used. These new applications, such as in situ quantitative calcium and pH measurements, would provide compelling reasons for researchers to purchase our confocal microscope.